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Monomeric CRISPR-Cas9 nucleases are widely used for targeted genome editing but can induce unwanted off-target mutations with high frequencies. Here we describe dimeric RNA-guided FokI nucleases (RFNs) that can recognize extended sequences and edit endogenous genes with high efficiencies in human cells. RFN cleavage activity depends strictly on the binding of two guide RNAs (gRNAs) to DNA with a defined spacing and orientation substantially reducing the likelihood that a suitable target site will occur more than once in the genome and therefore improving specificities relative to wild-type Cas9 monomers. RFNs guided by a single gRNA generally induce lower levels of unwanted mutations than matched monomeric Cas9 nickases. In addition, we describe a simple method for expressing multiple gRNAs bearing any 5' end nucleotide, which gives dimeric RFNs a broad targeting range. RFNs combine the ease of RNA-based targeting with the specificity enhancement inherent to dimerization and are likely to be useful in applications that require highly precise genome editing.

The RNA-guided endonuclease Cas9 is a versatile genome-editing tool with a broad range of applications from therapeutics to functional annotation of genes. Cas9 creates double-strand breaks (DSBs) at targeted genomic loci complementary to a short RNA...

Although CRISPR-Cas9 nucleases are widely used for genome editing, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM). As a result, it can often be difficult to target double-stra...

CRISPR RNA-guided nucleases (RGNs) are widely used genome-editing reagents, but methods to delineate their genome-wide, off-target cleavage activities have been lacking. Here we describe an approach for global detection of DNA double-stranded breaks ...